Generally, cell sorting devices separate cell populations of interest from a suspension and/or other types of cells. The principal method of Operation of early cell sorting devices relied on a cell's physical parameters to distinguish that cell from a suspension and/or other types of cells. Examples of bulk cell sorting techniques include filtration, which is based on cell size, and centrifugation, which is based on cell density. These techniques are effective as long as the cell population of interest is significantly different, with respect to size or density, from the suspension and/or the other cells in the population (i.e. a separation of red blood cells from blood). However, when the cell population of interest does not differ significantly in size or density, the filtration and centrifugation techniques are ineffectual.
In attempting to address this problem, a technique was developed which did not rely on cell size or density differences relative to the suspension and/or the other cells in the population. This technique was based on the presence of a tagging element, which was attached to the surface of the cell. This tagging technique has evolved to become a significant analytical tool in basic biological studies, applied biological studies, in the clinical diagnosis of diseases and the rapidly developing cell-based therapies in the treatment of diseases.
One application of this tagging technique is known as Fluorescence-Activated Cell Sorting (hereinafter FACS). In the FACS technique, an antibody-fluorescent label conjugate is used to tag a specific cell surface marker. The primary mode of Operation of a FACS sorter is binary in nature, that is, it determines whether a cell has the threshold number of fluorescent labels (i.e. positive sorting) or it does not have the threshold number of fluorescent labels (i.e. negative sorting). This determination is made by passing cells, single file, through a device which can determine whether each cell includes the parameter of interest (e.g. fluorescence). The binary separation is determined by the setting of a threshold or “gate” (also sometimes called a trigger). While the value of this “gate” or trigger is adjustable (i.e. quantitatively), the sorting process is still binary based on the threshold setting.
Furthermore, the rate of cell separation is relatively slow due to the fact that FACS sorters operate by examining a single cell at a time. Generally, a FACS sorter can provide a cell sorting rate of up to 30.000 cells/second. Higher cell sorting rates are possible, however, these higher sorting rates may decrease the yield. Still further, FACS sorters are relatively expensive and thus, most laboratory facilities are equipped with a limited number of sorters.
Another technique employing cell tagging as a basis for separation is known as High Gradient Magnetic Separation (hereinafter HGMS). The concept of sorting materials based on their magnetic responsiveness was first introduced in the industrial and mining arts. These methods relied on the intrinsic magnetic properties of the sorted material (generally, iron (i.e. magnetic) from non-iron parts (i.e. non-magnetic)) as a basis of Operation.
More particularly, in HGMS, a heterogeneous cell population, which includes a cell sub-population having magnetic cell tags, is passed through a magnetic field. As the heterogeneous cell population passes through the magnetic field, the cell sub-population labeled with the magnetic cell tags, becomes magnetically responsive to the applied magnetic field. That is, the cell sub-population including the magnetic tags will be subjected to a magnetic force which will cause the cells to be either attracted to (in the typical case), or repelled from, the magnetic field's source. Typically, the cell sub-population having the magnetic tags is attracted to the source of the magnetic field and collected by adhering to the magnetic source itself, or adhering to a cell collector device situated near the magnetic source. Therefore, the primary mode of the HGMS is also binary in nature, that is, it determines whether a cell has the magnetic tags or not.
The HGMS system, however, also has several drawbacks. Firstly, the cell sub-population of interest can be physically damaged during the HGMS process because of their forced magnetic massing at the collector device. Secondly, because the HGMS process sorts cells on the same fundamental principle as the FACS system, the HGMS method is also binary in nature. That is, both the FACS and HGMS systems separate cells based on the presence or absence of a parameter of interest (i.e. fluorescence and magnetic responsiveness, respectively).
Conventional flow cell sorters, such as FACS, are designed to have a nozzle with or without a flow chamber and use the principle of hydrodynamic focusing with sheath flow to separate or sort biological material such as cells. In addition, most sorting instruments combine the technology of ink-jet writing and the effect of gravity to achieve a high sorting rate of droplet generation and electrical charging. Despite these advances, many failures of these instruments are due to problems in the flow chamber or the nozzle. For example, orifice clogging, particle adsorption and contamination in the tubing may cause turbulent flow in the jet stream. These problems contribute to the great variation in illumination and detection in conventional FACS devices. Another major problem is known as sample carryover, which occurs when remnants of previous specimens left in the channel back-flush into the new sample stream during consecutive runs. Although such systems can be sterilized between runs, it is costly, time consuming, inefficient, and results in hours of machine down time for bleaching and sterilization procedures.
Sterile cell sorting is one of the biggest challenges in flow cytometry. Usually cell sorter are not in a sterile environment and therefore the risk of contaminations exists. The ability to prepare a cell sorter for aseptic sorting is essential. Standard protocols for performing a sterile sort are based on washing procedures by using Ethanol or other sterilizing reagents and should be performed in a time-consuming daily routine. However, these reagents are expensive and some are toxic for cells if they are not completely washed.
It is described in the state of the art, that ultraviolet systems can be used for water disinfection.
Ultraviolet light is a technology used in the drinking water treatment industry for disinfection of microorganisms in water. Recently, antimicrobial devices such as UV toothbrush sanitizers and UV disinfection wands have been disclosed. As with chemical disinfectants, the extent of disinfection that comes about from UV treatment is a function of both the duration and intensity of treatment. Ultraviolet (UV) radiation or light is defined as that portion of the electromagnetic spectrum between x rays and visible light, i.e., between 40 and 400 nm. The UV spectrum is divided into Vacuum UV (40-190 nm), Far UV (190-200 nm), UV-C (200-280 nm), UV-B (280-320), and UV-A (320-400 nm). Artificial sources of UV radiation include high, medium, and low pressure mercury vapor lamps, halogen lights, high-intensity discharge lamps, deuterium lamps, fluorescent and incandescent sources, and some types of lasers (excimer lasers, nitrogen lasers, and third harmonic Nd:YAG lasers). UV-C is almost never observed in nature because it is absorbed completely in the atmosphere, as are Far UV and Vacuum UV. Germicidal lamps are designed to emit UV-C radiation because of its ability to kill bacteria, viruses, molds, and spores or at least to prevent their reproduction. UV-B is typically the most destructive form of UV radiation because it has enough energy to cause photochemical damage to cellular DNA, yet not enough to be completely absorbed by the atmosphere. UV-A is the most commonly encountered type of UV light. Most phototherapy and tanning booths use UV-A lamps.
Ultraviolet radiation is used to kill microorganisms, molds and fungus in various environmental applications. UV sterilization is used for air-purification systems, water purification, aquarium and pond maintenance, laboratory hygiene and food and beverage protection.
UV treatment generally takes place only inside a specialized UV exposure chamber. It is useful for targeted elimination of microorganisms in air and water. UV sterilization leaves no residual chemical or radiation in the air or water and is harmless to untargeted animals and plants. UV works well with waterborne pathogens. Water should be filtered prior to UV exposure to improve penetration and the sterilization effect. Sterilized microorganisms remain in the air or water.
Germicidal ultraviolet radiation is primarily intended for the destruction of bacteria and other microorganisms in the air or on exposed surfaces. Ultraviolet light kills microorganisms by damaging the DNA. UV radiation disrupts the chemical bonds that hold the atoms of DNA together in the microorganism. If the damage is severe enough, the bacteria cannot repair the damage and will die. Longer exposure to UV light is necessary to ensure complete kill-off of all microorganisms. Unlike chemical treatments, UV-treated air or water does not resist re-contamination. In order for ultraviolet light to kill bacteria, the rays must directly strike the microorganism. Microorganisms floating in the air or on an outer surface may easily be reached by the ultraviolet rays and, therefore, are readily destroyed. If however, the microorganisms are hidden below the surfaces of a material or are not in the direct path of the rays, they will not be destroyed.
The exposure to ultraviolet necessary to kill bacteria is the product of time and intensity. High intensities for a short period of time, or low intensities for a long period are fundamentally equal in lethal action on bacteria disregarding the life cycle of the bacteria.